In the late fifties and early sixties Mucorales strains, in particular Blakeslea trispora, Choanephora cucurbitarum and Phycomyces blakesleeanus have been studied in order to develop a fermentation process for the production of carotenoids. Wildtype strains of these fungi accumulate carotenoids, in particular .beta.-carotene. However, the amounts that can be obtained with pure cultures are insignificant for commercial production.
.beta.-carotene is a lipid-soluble yellow chemical with provitamin A and reportedly with anticancer activities. Various applications in the food, feed, cosmetics, chemicals and pharmaceutical industries are reported.
Several processes that increase the .beta.-carotene production are based on the co-cultivation of strains of opposite mating type, (+) or (-). The interaction between mycelia of opposite mating types leads the formation of trisporic acids and to enhanced .beta.-carotene accumulation. Co-Is cultivation of Blakeslea trispora strains of opposite mating type in a batch-type fermentation process was developed by Hesseltine and Anderson (U.S. Pat. No. 2,865,814 and U.S. Pat. No. 2,890,989). Up to pilot-plant scale the process looked a promising way to produce .beta.-carotene. .beta.-Ionone was added to stimulate .beta.-carotene production of the two strains within the fermentor. In later years this process was optimized by several industrial companies. Numerous other additives have been used to stimulate the .beta.-carotene accumulation. However, these developments no longer were pursued as it became clear that these processes could not compete on a cost-price level with chemical processes to manufacture .beta.-carotene on an industrial scale.
Nowadays only in the former USSR a plant is in use to produce .beta.-carotene from Blakeslea trispora. Agricultural waste-products are used as raw materials for the fermentation and the .beta.-carotene obtained is used as a feed-additive.
Meanwhile academic research focussed on the biochemistry and regulation of .beta.-carotene biosynthesis using the filamentous fungus Phycomyces blakesleeanus as a convenient model system. The synthesis of .beta.-carotene in Phycomyces is subject to strict metabolic regulation. Mutations of the regulatory genes may overcome this strict regulation. Classical mutation experiments resulted in the development of strains with a strongly increased .beta.-carotene productivity even in the absence of stimulatory factors. Wildtype Phycomyces strains in the dark contain about 50 .mu.g .beta.-carotene per gram dry weight of biomass. Regulatory single mutants have been found to contain up to 6 mg .beta.-carotene/g dry weight. Double mutants have been isolated which contain over 10 mg .beta.-carotene per gram dry weight.
Phycomyces blakesleeanus is a filamentous fungus with hyphae containing no transverse cell walls (septa), thus the whole mycelium may be viewed as a single collective cell or coenocyte containing millions of nuclei. Unlike other fungi the hyphae never fuse together. Therefore, fusion can only be achieved by using artificial methods such as protoplast fusion or microsurgery or by transplantation of structures of the sexual cycle (T. Ootaki in Phycomyces, E. Cerda-Olmedo and ED Lipson eds. (1987), CSH Laboratory Press, 345-349; T. Suarez et al., Ibid 351-353). Reproduction of the fungus can take place either through a sexual cycle involving zygospores and germspores or by vegetative reproduction (Phycomyces, E. Cerda-Olmedo and E. D. Lipson eds., (1987) CSH Laboratory Press, 2).
The germspores that are the meiotic products of the mating process of two strains of opposite mating type and the resulting mycelia are usually homokaryotic, i.e. all their nuclei are genetically identical, however a few heterokaryotic germspores are formed spontaneously even in normal crosses. As a consequence some of the germspores give rise to intersexual heterokaryons which contain a mixture of (+) and (-) nuclei.
Such heterokaryons are morphologically different from the normal mycelia. The mycelia show a bright color due to the increased accumulation of .beta.-carotene and characteristic structures, so-called pseudophores, are formed.
The maintenance and propagation of intersexual heterokaryons is difficult since the nuclei show a clear tendency to segregate leading to sectors of mycelia having nuclei of one mating type only. The construction of intersexual heterokaryotic strains, wherein both the nuclei contain a recessive lethal mutation, to minimize the segregation of nuclei, has been the strategy by which F. J. Murillo Araujo et al. (U.S. Pat. No. 4,318,987) have succeeded to construct superproducing strains. These strains which can be described as stabilized intersexual heterokaryons of deregulated mutants were found to produce .beta.-carotene up to 25 mg/g dry weight.
However, even with these recessive lethal mutations, controlling the nuclear ratio to a certain extent, the intersexual heterokaryotic strains are not optimally suited for .beta.-carotene production. These intersexual heterokaryotic strains grow much worse than wild type strains and moreover they seldom or never sporulate.